Platelet-rich, arterial thrombi mediate tissue infarction in stroke, peripheral vascular disease, and myocardial infarction. During thrombus formation, platelets secrete their granule contents. The most abundant platelet granule, the alpha-granule, contains adhesion molecules, coagulation factors, and vasoactive factors that contribute to thrombus propagation. We have used a permeabilized platelet secretory model to define a role for SNARE proteins in membrane fusion events leading to platelet alpha-granule secretion. The mechanisms by which agonist-induced stimulation of the platelet results in SNARE protein-mediated membrane fusion, however, remain largely unknown. We have found that phosphatidylinositol (4,5)-bisphosphate synthesis is required for alpha-granule secretion. These studies demonstrated a role for type II phosphatidylinositol 5-phosphate 4-kinase in alpha-granule secretion. The synthetic pathway responsible for the synthesis of phosphatidylinositol (4,5)-bisphosphate required for alpha-granule secretion, however, is poorly characterized. Experiments described in Specific Aim 1 of this proposal will determine the relative contributions of type I phosphatidylinositol 4-phosphate 5-kinase and type II phosphatidylinositol 5-phosphate 4-kinase in platelet alpha-granule secretion. These studies will also determine whether type I phosphatidylinositol 5-phosphate 4-kinase serves as a downstream effector of protein kinase C during platelet alpha-granule secretion. Phosphatidylinositol (4,5)-bisphosphate mediates both remodeling of the platelet actin cytoskeleton and secretion of alpha-granules. Experiments described in Specific Aim 2 will determine whether the actin cytoskeleton mediates the effects of phosphatidylinositol (4,5)-bisphosphate in stimulating platelet alpha-granule secretion. Experiments described in Specific Aim 3 will define the role of the actin cytoskeleton in directing SNARE protein complex formation during alpha-granule secretion. These studies will define a critical activation pathway required for alpha-granule secretion and reveal interactions between the actin cytoskeleton and platelet secretory machinery that are important for platelet alpha-granule secretion.